SAFETY FEATURES IN THE DESIGN, MANUFACTURE AND CLINICAL MONITORING OF LENTIVECTORS FOR THE TREATMENT OF PARKINSON’S DISEASE, PROSTATE CANCER AND AIDS

SAFETY FEATURES IN THE DESIGN, MANUFACTURE AND CLINICAL MONITORING OF LENTIVECTORS FOR THE TREATMENT OF

PARKINSON’S DISEASE, PROSTATE CANCER AND AIDS.

FDA/BRMA 25-26 Oct 2001

Susan M KingsmanSenior Vice President

Research

The RetroviridaeThe Retroviridae

SpumavirusMammalian

C-typeMammalian

C-typeLentivirusLentivirus

D-type B-typeAvianC-type

BLV/HTLV

Most VectorsMost Vectors

All share RT(pol) but differ in morphology, pathogenicityand replication and gene expression characteristicsAll share RT(pol) but differ in morphology, pathogenicityand replication and gene expression characteristics

Equine Infectious Anaemia Virus, EIAV: A non-primate lentivirus Human Immunodeficiency Virus, HIV: A primate lentivirusEquine Infectious Anaemia Virus, EIAV: A non-primate lentivirus Human Immunodeficiency Virus, HIV: A primate lentivirus

SIVMNDSIVCPZ

SIVSMMH4

HIV-2ROD

CAEV

BIV

EIAV

HIV-1MN

FIVPPR

FIV14

VLVOMVVSA

SIVAGMTYO

SIVAGM677

Lentiviruses: Analysis of Pol SequencesLentiviruses: Analysis of Pol Sequences

PrimateLentiviruses

Non-PrimateLentiviruses

Why Develop Lentivirus-Based Vectors?Unique Advantages for Gene Therapy

Why Develop Lentivirus-Based Vectors?Unique Advantages for Gene Therapy

• Vectors for long term, stable therapy of chronic diseases• Vectors for unmet medical needs• Vectors for long term, stable therapy of chronic diseases• Vectors for unmet medical needs

1. Simplicity

2. Up to 11kb capacity

3. Defined integration of genes

4. Long term expression

5. Transduction of non-dividing cells

6. Transduction of slowly dividing cells

Hippocampus/septal cholinergic system:Site of learning & memory Major damage in strokePrimary target in Alzheimer’s

Substantia nigra:Primary target in Parkinson’s

Striatum:Primary target in Huntington’s

Nucleus acumbens:site of addictions

Spinal motoneurons:Primary target inALS, SMN

Muscle

EIAV Vectors: Gene Transfer to the Nervous SystemEIAV Vectors: Gene Transfer to the Nervous System

A powerful approach to the management of neurological diseaseA powerful approach to the management of neurological disease

Rat

EIAV Vectors: Long Term Gene Expression in Vivo

EIAV Vectors: Long Term Gene Expression in Vivo

8 days

24 weeks

Sustained expression minimises the need for repeat invasive deliverySustained expression minimises the need for repeat invasive delivery

Rat thalamus

• Intramyocardial (107)• 80 rats• 14d, no clinical signs of toxicity

• Intra-cranial (107)• Over 100 rats• Up to 8mths• No clinical signs• No histological abnormality• Mild acute inflammation

EIAV Vectors: No Systemic Toxicity at Doses Relevant to the Clinic

EIAV Vectors: No Systemic Toxicity at Doses Relevant to the Clinic

•Tail vein (107)•12 rats 21d•No liver tox or damage by histology•No vector in liver or lung

Following MLV toxicity and biodistribution protocolsFollowing MLV toxicity and biodistribution protocols

• Intra-tumoral (107) • 21d No abnormal tox

CMV

Tonic expression of dopamine, augmentation of L-DOPA therapyTonic expression of dopamine, augmentation of L-DOPA therapy

An EIAV-Based Vector for The Treatment of Parkinson’s Disease

An EIAV-Based Vector for The Treatment of Parkinson’s Disease

IRES SIN LTR

TH CH1 AADCCMVp

IRES

Tyrosine Hydroxylase(TH)

L-DOPA

Dopamine

L-Tyrosine

GTP-Cyclohydrolase 1(CH1)

BH4

Aromatic AADopa Decarboxylase (AADC)

6-OHDA lesion

Control

An EIAV Based Vector for the Treatment of Late Stage Prostate Cancer

An EIAV Based Vector for the Treatment of Late Stage Prostate Cancer

•Slow growing tumour •100 fold improved TK•TK clinical experience•Prodrug strategy terminates therapy easily•Therapy selective for dividing cells•Recurrent intractable malignant disease•Local delivery with transrectal ultrasound guidance

• MCA Preliminary discussions on LentiVectors• GTAC and MCA protocol outline early 2002• MCA Preliminary discussions on LentiVectors• GTAC and MCA protocol outline early 2002

LNCaP

Efficacy in progress

PC-3 xenograftAb

s

0

20

40

60

80

100

120

% C

on

tro

l

GFP -TKm

Wst-1 assay/PC3

0

10

1

0.2

[GCV]

PUROCMVp TKmCMVp

LentiVector SpecificationLentiVector Specification

Basic Designand Production

Enhanced Designand Production

Gene specific considerations

Disease and patient specific considerations

Growth factorsProdrug activating enzymes

Metabolic replacement Cell death regulators

Terminal disease Chronic disease

Adults Children

Specification is an on-going process. Clinical evaluation should occurin parallel with vector and process development for certain indicationsSpecification is an on-going process. Clinical evaluation should occurin parallel with vector and process development for certain indications

LentiVectors : Key Vector-Specific Safety Issues


•Replication Competent Lentiviruses (RCL) in the product

Minimise the generation and impact of RCLs

•Mobilisation of the transfer vector in target cells

Minimise the inappropriate dissemination of the transgene

Minimising the Generation and Impact of RCLs


• If possible use a non-pathogenic virus

• Split the vector production system into at least three components

• If possible use a stable producer cell line

• Eliminate all non-essential coding and cis-active sequences

• Minimise the potential for homologous recombination

• Reduce packaging of vector helper components

• Use sensitive calibrated assays for detecting RCLs

If possible use a non-pathogenic virus

Consequences of any RCL may be minimisedConsequences of any RCL may be minimised

Minimising the Generation and Impact of RCL


Features of HIV and EIAV Features of HIV and EIAV

HIV• Extensive analysis • Complex (6 accessory genes)• Human pathogen• Replicates in human cells• Fatal immunodeficiency• Global pandemic• 30M AIDS/HIV+ve (worldwide)

EIAV• Moderate analysis• Simple (3 accessory genes)• Equine pathogen• No replication in human cells• Self limiting anaemia• Endemic in horses in the Tropics• Rare disease in stables (worldwide)

• Different lentiviruses have different safety profiles• EIAV is rarely fatal in horses, does not cause immunodeficiency• No a priori expectation that EIAV would be a human pathogen• Low probability of a patient encountering EIAV

• Different lentiviruses have different safety profiles• EIAV is rarely fatal in horses, does not cause immunodeficiency• No a priori expectation that EIAV would be a human pathogen• Low probability of a patient encountering EIAV

Split the vector production systeminto at least three components

Increases the number of recombination events required to generate an RCLIncreases the number of recombination events required to generate an RCL



LentiVectors: The Basic SystemLentiVectors: The Basic SystemU3 R U5 U3 R U5

gag-pol envRNA1 2 3

3 ComponentSplit System

Vector genomeU3 R U5Therapeutic Gene(s)U3 R U5

gag-polp Gag-pol packaging plasmid

envp Env packaging plasmid

Low probability of generating RCLs requiring four cross overs, two non-homologousLow probability of generating RCLs requiring four cross overs, two non-homologous

2

3

1

If possible use stable producer cell lines



• DNA recombination unlikely• Genetically stable• Conventional manufacturing parameters established• Characterised starting materials

• DNA recombination unlikely• Genetically stable• Conventional manufacturing parameters established• Characterised starting materials

Eliminate all non-essential coding and cis-active sequences

Reduces recombination and pathogenic potentialReduces recombination and pathogenic potential



Lentiviral ProViral GenomesLentiviral ProViral Genomes

Transfer vector construction is complicated due to multiple introns and accessory genesTransfer vector construction is complicated due to multiple introns and accessory genes

EIAV: Non-Primate, 3 accessory genes

SD

LTRgag polS2

envrev revtatLTR

RT RH DU IN

tat

cPPT

RRE Pun

TAR

RRE

HIV: Primate, 6 accessory genes

LTR

SD

gagvpr

vpu

vif

pol

RT RH INLTRnef

tat

rev

env

Pun

TAR

cPPT

The ‘Additional’ Genes of LentivirusesThe ‘Additional’ Genes of Lentiviruses

+ = Tat, Nef, Vif, Vpu, Vpr, potential pathogenicity factors in man+ = Tat, Nef, Vif, Vpu, Vpr, potential pathogenicity factors in man

Tat

Rev

Nef

Vpu

Vpr

Vif

S2

Binds TAR RNA sequence, activates transcription, for HIV-1 Tat, possible growth factor and oncogene via anti-apoptotic effects

Binds RRE RNA, promotes stability/transport of Gag-pol and Env RNAs. No pathogenic properties to date

Down regulates CD4 and plays a role in early stages of infection

CD4 degradation and virus release

Transport of PIC to nucleus and cell cycle arrest

Cell dependent virus assembly factor

Enhances pathogenicity in horses

EIAV HIV

+ +

+ +

++++

+

1st Generation EIAV Vector Producer Cell:8Z-201st Generation EIAV Vector Producer Cell:8Z-20

Issues: Recombination homology, S2 and Rev expressed in productionIssues: Recombination homology, S2 and Rev expressed in production

John Olsen’sBiG-45PackagingLine pTOG + pcDNA6/TR

pEV53B (Expresses gag/pol, S2, Rev)

VSV-GTetO2

CMVp

pA

TetRCMVp

pA

pA

Gag PolRev Rev

S2

SD

CMVpCMVp

RRE

*

pONY8.0Z (Some EIAV coding regions present but non-expressing)

*Mut

LTR

Lac ZCMVp

*

**

SDTAR

CMVpCMVp*

RRE Pu

*

VIRUS YIELD FROM 8Z-20 PRODUCER

VIRUS YIELD FROM 8Z-20 PRODUCER

1x10 6

Time (hours)

1x10 5

1x10 4

1x10 3

Viral Titer (TU/ml)

0 -24 24 -48 48 -72 96 -120 72 -96

Sodium butyrate

Doxycycline

+ - - - -

+ + + + +

•Stable producer line constructed•Scaled in roller bottles >106 t.d.u./ml for 5 days•Standard vector for benchmarking improvements

•Stable producer line constructed•Scaled in roller bottles >106 t.d.u./ml for 5 days•Standard vector for benchmarking improvements

EIAV Minimal Transfer VectorEIAV Minimal Transfer Vector

• Minimal transfer vectors do not express any EIAV proteins. • >8kb insert capacity. • Homology at • cPPT and RRE optimise yields or potency but create regions of homology

• Minimal transfer vectors do not express any EIAV proteins. • >8kb insert capacity. • Homology at • cPPT and RRE optimise yields or potency but create regions of homology

pONY8.1 series: GFP, LacZ, (optional cPPT, WPRE, RRE)

pEV53B

RRE

pARev Rev

Pol

S2CMVp

SD

*

Gag

RRE

WPRE

cPPTLac Z LTR

CMVpCMVp

cPPT

Minimise the potential for homologous recombination

• Remove all homologous sequences from the Gag-Pol packaging plasmid• Do not compromise expression of Gag-Pol• Examine requirement for RRE/Rev

• Remove all homologous sequences from the Gag-Pol packaging plasmid• Do not compromise expression of Gag-Pol• Examine requirement for RRE/Rev



pEV53B

pARev Rev

Pol

S2CMVp

SD

*

Gag

cPPT RRE

Lentivirus Codon UsageLentivirus Codon UsageMH WT CO MH WT CO MH WT CO MH WT CO

Ala A 13 46 8 Cys C 68 10 70 Leu A 3 14 3 Ser C 34 35 55GC C 53 19 65 TG T 32 90 30 CT C 26 8 17 AG T 10 10 3

G 17 11 8 G 58 14 70 TC A 5 38 17

T 17 24 19 Gln A 12 53 21 T 5 11 6 C 28 10 14

CA G 88 47 79 TT A 2 42 6 G 9 3 7

Arg A 10 58 10 G 6 11 0 T 13 3 3

AG G 18 29 11 Glu A 25 65 38

CG A 6 6 0 GA G 75 35 62 Lys A 18 58 28 Thr A 14 45 16

C 37 0 61 AA G 82 42 72 AC C 57 29 52G 21 6 10 Gly A 14 53 21 G 15 0 19

T 7 0 5 GG C 50 21 55 Phe C 80 45 45 T 14 26 13

G 24 24 24 TT T 20 55 55

Asn C 78 29 71 T 12 3 0 Tyr C 74 20 80AA T 22 71 29 Pro A 16 52 24 TA T 26 80 20

His C 79 30 90 CC C 48 15 39

Asp C 75 64 70 CA T 21 70 10 G 17 3 21 Val A 5 56 4

GA T 25 36 30 T 19 30 15 GT C 25 8 20

Ile A 5 58 8 G 64 24 76AT C 18 19 92 T 7 12 0

T 77 23 0

Lentiviruses maintain a suboptimal codon biasLentiviruses maintain a suboptimal codon bias

Codon Optimised Gag-PolCodon Optimised Gag-Pol

• Codons changed across gag-pol except in the critical frame-shifting overlap region• Removes all blocks of sequence homology• Rev-independent• Achieved for EIAV and HIV gag-pol

• Codons changed across gag-pol except in the critical frame-shifting overlap region• Removes all blocks of sequence homology• Rev-independent• Achieved for EIAV and HIV gag-pol

pESYNGP

pEV53B

RRE

pARev Rev

Pol

S2CMVp

CMVp

pAGag Pol

SD

*

Gag

cPPT

Minimal EIAV Vector SystemMinimal EIAV Vector System

• No obligate requirement for any accessory genes but Rev/RRE can improve yields• No functional viral proteins or significant coding regions in the transfer vector• Transfer vector contains only 1100 nt of original EIAV nucleic acid• No significant homology between components• Approved in UK by HSE for CL1 use with non-toxic genes i.e. lowest containment• Similar HIV-1 system has been constructed at OBM

• No obligate requirement for any accessory genes but Rev/RRE can improve yields• No functional viral proteins or significant coding regions in the transfer vector• Transfer vector contains only 1100 nt of original EIAV nucleic acid• No significant homology between components• Approved in UK by HSE for CL1 use with non-toxic genes i.e. lowest containment• Similar HIV-1 system has been constructed at OBM

pTOG

pESYNGP

VSV-GTetO2

pACMVp

CMVp

pAGag Pol WPRE

cPPTLac Z LTR

CMVpCMVp

RRE

cPPT

pONY8.1

2nd Generation Packaging Cell:ESYN-29-iG Series

2nd Generation Packaging Cell:ESYN-29-iG Series

pTOG + pcDNA6/TR

VSV-GTetO2

CMVp

pA

TetRCMVp

pA

pTO-Rev

RevTetO2

CMVp

pAOptional codon optimised Rev

pESYNGP

Work in progress in collaboration with John Olsen: The system aims to meet the 8Z-20 production characteristics Work in progress in collaboration with John Olsen: The system aims to meet the 8Z-20 production characteristics

CMVp

pAgag pol

Reduce packaging of vector helper components

Lessons from retroviral vectors: Eliminate packaging and dimerisation signalsLessons from retroviral vectors: Eliminate packaging and dimerisation signals



XX

Self Packaging Gag-Pol TranscriptsSelf Packaging Gag-Pol Transcripts

Gag-PolCMV

CMV

STOP

GagCMV

WT

Gag-Pol expression plasmids

(various)

(Synthetic)

RNA only control

U3 R U5 Gag-PolPBS

• Defining sequences that allow Gag-Pol proteins to package the cognate mRNA• Defining sequences that allow co-packaging of Gag-Pol and transfer vector RNA• Defining sequences that allow Gag-Pol proteins to package the cognate mRNA• Defining sequences that allow co-packaging of Gag-Pol and transfer vector RNA

Gag-Pol

Self-packaging of EIAV Gag-Pol mRNASelf-packaging of EIAV Gag-Pol mRNA

Partial or complete removal of reduces packaging to background levelsNo evidence for ‘piggy backing’ via dimerisation with the transfer vectorPartial or complete removal of reduces packaging to background levelsNo evidence for ‘piggy backing’ via dimerisation with the transfer vector

1.0E-07

1.0E-06

1.0E-05

1.0E-04

1.0E-03EIAV Vector RNAActin

EIAV Gag-pol RNA

Actinbaseline

Gag-Pol

PackagedRNA

WT WT

Plus Transfer Vector

VLP-RNA Cell RNA



The assays must relate to the properties of any RCLThe assays must relate to the properties of any RCL

Use sensitive, calibrated assays to detect RCLs

R-U5 U3-REnv(v)gag-pol(e)

R-U5 U3-Rgag-pol(v) Env(v)

R-U5 U3-Rgag-pol(v) Env(e)

• All RCL’s have Gag-Pol• The most likely Gag-Pol is vector derived• Any other Gag-Pol is only rendered transmissible with

vector derived Env

Theoretical RCL structures:A Generic View

Theoretical RCL structures:A Generic View

• Screen for transmissible reverse transcriptase (Pol).• Qualify ambiguous results by a second screen for trasmissible Gag and Pol and

by screening for transmissible Env

• Screen for transmissible reverse transcriptase (Pol).• Qualify ambiguous results by a second screen for trasmissible Gag and Pol and

by screening for transmissible Env

Proposed Testing for RCLProposed Testing for RCL

• Test vector preparations and post-production cells following current CBER guidelines for MLV derived vectors

• Use F-PERT assay as the primary assay tool Fluorescence-based Product Enhanced Reverse Transcriptase Assay

• Use PCR assay to resolve ambiguous PERT assay results Specific assay for hypothetical recombinant molecular structures

We have developed these assays and strategies in collaboration and

discussion with relevant UK Government agencies (LGC, NIBSC) and with

a contract manufacturer, Q-One Biotech

We have developed these assays and strategies in collaboration and

discussion with relevant UK Government agencies (LGC, NIBSC) and with

a contract manufacturer, Q-One Biotech

F-PERT: Product Enhanced Reverse Transcriptase Assay

(MLV, AMV, HIV-1/2, EIAV, SRV-1, HTLV-1/2, FLV, FIV, BIV, CAEV, MVV, PoERV, SFV, SMRV)

F-PERT: Product Enhanced Reverse Transcriptase Assay

(MLV, AMV, HIV-1/2, EIAV, SRV-1, HTLV-1/2, FLV, FIV, BIV, CAEV, MVV, PoERV, SFV, SMRV)

ABI Prism 7700(TaqMan)

pol(10-100/virion)

Disrupted VLP

Reverse transcription of

MS2 RNA

NP-40MS2 phage RNA

RT primerPol

MS2 cDNA

• Independent of the nature of events leading to RCLs • Broadly applicable and high sensitivity (10-100 particles)• Modified to control against false positives

• Independent of the nature of events leading to RCLs • Broadly applicable and high sensitivity (10-100 particles)• Modified to control against false positives

1012141618202224262830323436384042

MLV n

eat

MLV-1

MLV-2

MLV-3

MLV-4

MLV-5

EIAV n

eat

EIAV-1

EIAV-2

EIAV-3

EIAV-4

EIAV-5

MLV u

ndisru

pted

EIAV u

ndisru

pted

Med

ia

Med

ia/D

B

water

/DB

water

MS2c

DNA

Ct

The F-PERT Assay Is Highly SensitiveThe F-PERT Assay Is Highly Sensitive

Both MLV (Mn2+-dependent) and EIAV (Mg2+-dependent) RT activities were detected by PERT assay over a wide range of dilutions.Both MLV (Mn2+-dependent) and EIAV (Mg2+-dependent) RT activities were detected by PERT assay over a wide range of dilutions.

ESYNGP ORF (4658 bps)

1000 2000 3000 4000

GAG

GAG E POSGAG I POS

GAG I NEG

POL

GAG E NEG

POL E POSPOL I POS

POL I NEGPOL E NEG

VSV-G ORF (1650 bps)

500 1000 1500

VSV E POS

VSV I POSVSV I NEG

VSV E NEG

PCR Assay: Nested Primers For The Detection Ofgag, pol and VSV-G

PCR Assay: Nested Primers For The Detection Ofgag, pol and VSV-G

• Nested primers detect gag, pol and env, separately or as a linkage group• No cross reaction with other retroviruses or lentiviruses• Developed in collaboration with the Laboratory of the Government Chemist

• Nested primers detect gag, pol and env, separately or as a linkage group• No cross reaction with other retroviruses or lentiviruses• Developed in collaboration with the Laboratory of the Government Chemist

VSV-G

100 510 1 0.1 0.01

M +ve -ve 1 -ve2

50

copy number

gag

pol

target sensitivityCOPIES/5 X 105

genomes

1-10

1-10

1-10

Sensitivity established in the context of assay cell genomic DNA Sensitivity established in the context of assay cell genomic DNA

Sensitive PCR Detection of all Vector ComponentsSensitive PCR Detection of all Vector Components

Transduction by test article Split cells at appropriate ratio

over appropriate time

PERT assay on cell-free supernatants

PCR assay for EIAV gag, pol and env on genomic DNA

• Amplification process is essential• Readily adapted for screening producer cells • Amplification process is essential• Readily adapted for screening producer cells

RCL Amplification and DetectionRCL Amplification and Detection

Positive StandardFeLV B/C•Efficient amplification in 293 & lymphoid cells• Regulatory standard• No LentiVector interference

HEK293•Tranduced by wide range of Vectors and pseudotypes

Lymphoid (Raji, H9, Sup1) • Potential in-patient target for RCLs

• If possible use a non-pathogenic virus

• Split the vector production system into at least three components

• Preferably use a stable producer cell line

• Eliminate all non-essential coding and cis-active sequences

• Minimise the potential for homologous recombination

• Reduce packaging of vector helper components

• Use sensitive, calibrated assays for RCLs after amplification in human cells



Oxford BioMedica proposes that this is a specification for discussionOxford BioMedica proposes that this is a specification for discussion



•Replication Competent Lentiviruses in the product




Minimising MobilisationMinimising Mobilisation

Use a transfer vector that is poorly mobilised

Reduce the level of mobilisable RNA in the target cell


HIV-1 is the agent for mobilisation that is of most concernHIV-1 is the agent for mobilisation that is of most concern

Use a transfer vector that is poorly mobilised

LentiVectors: Cross - Packaging AssaysLentiVectors: Cross - Packaging Assays

HEK293T

gag-polGFPEIAV

HIV

MLV

gag-polGFP

gag-polGFP

Gag-Pol

GFP Biological titre on D17 cellsby FACS for GFP after 5dAnd after 2 serial passages

= 1

= 1

= 1

1/4

1/4

Test pairwise combinations for packaging and transductionTest pairwise combinations for packaging and transduction

Virion associatedGFP RNA(TaqMan)

Env

LentiVector and Retroviral Vector Cross Transmission: Initial TitresLentiVector and Retroviral Vector Cross Transmission: Initial Titres

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

EIAV HIV MLV

Titr

e (

TU

pe

r m

l)

Gag-Pol

EIAV genome

MLV genome

HIV genome

• Cross mobilisation between EIAV and HIV occurs at less than 1000 fold the efficiency of homologous mobilisation• EIAV and MLV are similarly poorly mobilised by HIV• NB: MLV is already in the clinic in HIV+ve patients

• Cross mobilisation between EIAV and HIV occurs at less than 1000 fold the efficiency of homologous mobilisation• EIAV and MLV are similarly poorly mobilised by HIV• NB: MLV is already in the clinic in HIV+ve patients


Reduce the level of mobilisable RNA in the target cell

• Achieved by modifying the transfer vector e.g. SIN vectors• Should not be required for LentiVectors that are already poorly mobilised• Achieved by modifying the transfer vector e.g. SIN vectors• Should not be required for LentiVectors that are already poorly mobilised

Self Inactivating (SIN) EIAV VectorsSelf Inactivating (SIN) EIAV Vectors

pONY8.1 SIN Vector: Configuration in the transduced target cell

Very low level expression of genomic RNA

• Residual viral genome is 900nt• Deletes the LTR: no evidence that this deletion is beneficial per se and it reduces• vector options for second transcription units.• SIN transfer vectors cannot be introduced into packaging cells by transduction unless a conditional SIN is used

• Residual viral genome is 900nt• Deletes the LTR: no evidence that this deletion is beneficial per se and it reduces• vector options for second transcription units.• SIN transfer vectors cannot be introduced into packaging cells by transduction unless a conditional SIN is used

mRNA in target cell

CMVp*

SIN LTR

LacZ

In progress: SIN and EIAV LTR vectors reduce transferable RNA levels in the target cell by at least 3 logs. In progress: SIN and EIAV LTR vectors reduce transferable RNA levels in the target cell by at least 3 logs.

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

copies per actin units 5 8,742 11,133 35,116,825

SINControl LTR CMV

8.1SIN 8.0 LTR

8Z20CMV

Performance of EIAV LTR and SIN VectorsPerformance of EIAV LTR and SIN Vectors

Mobilisation of HIV Vectors by HIVMobilisation of HIV Vectors by HIV

A special case for the treatment of AIDS?

Turns virus escape into an attributeTurns virus escape into an attribute

ImmStat: A Mobilisable Therapeutic Rz Vector for the treatment of AIDS ImmStat: A Mobilisable Therapeutic Rz Vector for the treatment of AIDS

• pSYNGP allows Rz directed against the conserved gag-pol• No immune responses anticipated against therapeutic RNA• Mobilisation amplifies the therapy if virus escapes the first wave of Rzs

• pSYNGP allows Rz directed against the conserved gag-pol• No immune responses anticipated against therapeutic RNA• Mobilisation amplifies the therapy if virus escapes the first wave of Rzs

Tat inducible genomic and therapeutic RNA

ImmStat Vector Particle Constitutive therapeutic RNA

Anti-HIVribozymes

HIV genome

Anti-HIV and chemokine receptor Ribozymes

CMV R-U5 Multi-Rz CMVp LTR

HIV-1 Production from Infected ImmStatPositive and Negative cells

HIV-1 Production from Infected ImmStatPositive and Negative cells

0

5000

10000

15000

20000

25000

30000

0 2 4 6 8 10 12 14 16 18 20 22 24

Days

RT

Un

its

HIVGFP

HIVGFP- ImmStat

ImmStat delays HIV replication ImmStat delays HIV replication

? Genetic variant ?



•Replication Competent Lentiviruses in the product




A LentiVector System With a Good Safety Profile

A LentiVector System With a Good Safety Profile

pONY8.1 Z or G

pTOG

pESYNGP

VSV-GTetO2

pACMVp

pA

• No pathogenic proteins• Very poorly mobilised by HIV (sim. MLV)• No potential for homologous recombination• RCLs extremely unlikely

• No pathogenic proteins• Very poorly mobilised by HIV (sim. MLV)• No potential for homologous recombination• RCLs extremely unlikely

CMVp

gag pol

WPRE

cPPTLac Z LTR

CMVpCMVp

RRE

LentiVector Specification: A ProposalLentiVector Specification: A Proposal• A LentiVector system must show efficacy in a relevant animal model• It must be capable of scale production at GMP• It should be configured with the following points in mind

1. Eliminate non-essential proteins and sequences.

2. Ensure extremely low, preferably zero, homologous recombination potential between vector components.

3. Show significant differential between self mobilisation and mobilisation with HIV or, use a vector that after integration generates a significant reduction in mobilisable RNA. Use MLV as a benchmark for mobilisation.

4. Use a minimum of three split components in a stable packaging cell lineor in a very low-homologous-recombination transient system.

5. Use sensitive, calibrated assays for RCLs (follow MLV guide) with an amplification step in an appropriate human cell line.

6. Test for no significant acute toxicity in the animal efficacy model.

V.N. Kim et al (1998) Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1. J. Virol. 72, 811

K.A. Mitrophanous et al (1999) Stable gene transfer to the nervous system using a non-primate lentiviral vector

E. Kotsopoulou et al (2000) A Rev-independent human immunodeficiencyvirus type 1 –based vector that exploits a codon-optimised gag-pol gene.J. Virol. 74, 4839

N.D. Mazarakis et al (2001) Rabies virus pseudotyping of lentiviral vectorsenables retrograde axonal transport and acccess to the nervous system after peripheral delivery. Human Molecular Genetics, 10, 2109.

E.Martin-Rendon, et al (2001) Lentiviral vectors for the treatment of Neurodegenerative diseases. Curr. Opinion Molecular Therapeutics, 3, 476.

Supporting ReferencesSupporting References

The LentiVector TeamThe LentiVector Team

Virology

Kyri MitrophanousJonathan RohllFiona EllardFraser WilkesAnna OlsenPippa RadcliffeJames MiskinDanny ChipchaseMargaret Esapa

Neurobiology

Nick MazarakisEnca Martin-RendonMimoun AzzouzKaren VincentRob Barber

Production

Andrew Slade Wendy BlakemoreJay BrownLinda Dove

Collaborators

John Olsen Karen O Malley James UneyLGCNIBSCQ-One Biotech

BioMedica Inc

Doug JollyHolger Roehl

Consultants

Jonathon WeberMark SaundersRobin WeissMary CollinsPatrick AebischerKrys Bankiewicz

Biological Systems

Stuart NaylorKatie BinleyOn KanLeigh GriffithsEmma CarterZoe Askham

Documents

SAFETY FEATURES IN THE DESIGN, MANUFACTURE AND CLINICAL MONITORING OF LENTIVECTORS FOR THE TREATMENT OF PARKINSON’S DISEASE, PROSTATE CANCER AND AIDS